Intranasal infection with vaccinia virus co-expressing interferon epsilon (VV-HIV-IFN-ε) was used to evaluate the role of IFN-ε in mucosal immunity. VV-HIV- IFN-ε infection induced a rapid VV clearance in lung that correlated with (i) an elevated lung VV-specific CD8(+)CD107a(+)IFN-γ(+) population expressing activation markers CD69/CD103, (ii) enhanced lymphocyte recruitment to lung alveoli with reduced inflammation, and (iii) an heightened functional/cytotoxic CD8(+)CD4(+) T-cell subset (CD3(hi)CCR7(hi)CD62L(lo)) in lung lymph nodes. These responses were different to that observed with intranasal VV-HA-IFN-α(4) or VV-HA-IFN-β infections. When IFN-ε was used in an intranasal/intramuscular heterologous HIV prime-boost immunization, elevated HIV-specific effector, but not memory CD8(+)T cells responses, were observed in spleen, genito-rectal nodes, and Peyer's patch. Homing marker α4β7 and CCR9 analysis indicated that unlike other type I IFNs, IFN-ε could promote migration of antigen-specific CD8(+)T cells to the gut. Our results indicate that IFN-ε has a unique role in the mucosae and most likely can be used to control local lung and/or gut infections (i.e., microbicide) such as tuberculosis, HIV-1, or sexually transmitted diseases.